- Volume 148, Issue 8, 2002
Volume 148, Issue 8, 2002
- Review Article
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- Microbiology Comment
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- Research Paper
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Prospecting for novel lipase genes using PCR a
aThe GenBank accession number for the sequence reported in this paper is AF421484.
A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described. The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design. Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA. This lipase showed less than 20% similarity with other known lipases at the amino acid level. The study also revealed that distantly related members of the α/β hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.
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Evidence for protection of nitrogenase from O2 by colony structure in the aerobic diazotroph Gluconacetobacter diazotrophicus
Gluconacetobacter diazotrophicus is an endophytic diazotroph of sugarcane which exhibits nitrogenase activity when growing in colonies on solid media. Nitrogenase activity of G. diazotrophicus colonies can adapt to changes in atmospheric partial pressure of oxygen (pO2). This paper investigates whether colony structure and the position of G. diazotrophicus cells in the colonies are components of the bacterium’s ability to maintain nitrogenase activity at a variety of atmospheric pO2 values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO2 of 2 and 20 kPa. Imaging of live, intact colonies by confocal laser scanning microscopy and of fixed, sectioned colonies by light microscopy revealed that at 2 kPa O2 the uppermost bacteria in the colony were very near the upper surface of the colony, while the uppermost bacteria of colonies cultured at 20 kPa O2 were positioned deeper in the mucilaginous matrix of the colony. Disruption of colony structure by physical manipulation or due to ‘slumping’ associated with colony development resulted in significant declines in nitrogenase activity. These results support the hypothesis that G. diazotrophicus utilizes the path-length of colony mucilage between the atmosphere and the bacteria to achieve a flux of O2 that maintains aerobic respiration while not inhibiting nitrogenase activity.
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Functional analysis of microbial communities in aerobic–anaerobic sequencing batch reactors fed with different phosphorus/carbon (P/C) ratios
More LessFluorescence in situ hybridization (FISH) was used to analyse the community composition of a sequencing batch reactor (SBR) operating with aerobic–anaerobic cycling and fed acetate as its sole carbon source. Phosphorus was removed from the SBR microbiologically. Marked shifts in the community structure occurred as the phosphorus/carbon (P/C) ratio in the feed was changed. When the P/C ratio was shifted from 1:10 to 1:50, FISH analysis showed that the percentage of β-Proteobacteria fell from ca 77% of the total bacteria to ca 38%. This decrease in the β-Proteobacteria coincided with a reduction in both the proportions of the β-proteobacterial Rhodocyclus-related phosphorus-accumulating bacteria and the biomass phosphorus content. FISH/microautoradiography and FISH/poly β-hydroxyalkanoate (PHA) staining showed that the Rhodocyclus-related bacteria assimilated acetate and synthesized PHAs anaerobically, and that they accumulated phosphorus aerobically. No Acinetobacter spp. could be detected in any of the communities, casting further doubt on their role in phosphorus-removing activated sludge systems. As the feed P/C ratio decreased there was a corresponding increase in the proportion of α-Proteobacteria and, to a smaller extent, in the proportion of γ-Proteobacteria; both the α- and γ-Proteobacteria consisted mostly of tetrad-forming cocci, fitting the description of the so-called ‘G-bacteria’ morphotype. The change in the proportions of Proteobacteria present paralleled increases in the biomass glycogen content. Both the α- and β-proteobacterial ‘G-bacterial’ populations assimilated acetate and synthesized PHA anaerobically. The α-Proteobacteria are considered responsible for glycogen production in these SBR systems.
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Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal c
More LesscThe EMBL accession numbers for the sequences reported in this paper are X84472 (strain SBR1029 16S rDNA), X84474 (strain SBR1031 16S rDNA), X84498 (strain SBR1064 16S rDNA), X84565 (strain SBR2022 16S rDNA), X84576 (strain SBR2037 16S rDNA) and X84607 (strain SBR2076 16S rDNA).
Most filamentous bacteria in biological nutrient removal (BNR) processes have not been identified beyond their morphotype and simple staining reactions. Furthermore, the majority of sludge filaments observed under the microscope do not hybridize to commonly used phylogenetic probes for well characterized bacterial phyla such as the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Specific 16S rRNA-targeted oligonucleotide probes were designed for the phylum Chloroflexi (green non-sulfur bacteria) and optimized for use in fluorescence in situ hybridization. Chloroflexi have been implicated in BNR systems by phylogenetic identification of filamentous bacteria isolated by micromanipulation from sludge and culture-independent molecular phylogenetic surveys. The predominant morphotype responding to the probes was filamentous and these filaments were generally abundant in 10 Australian full-scale and two laboratory-scale BNR samples examined. Filamentous bacteria responding to a subdivision 1 Chloroflexi probe were rare in the samples, whereas subdivision 3 Chloroflexi filaments were very common in some sludges. This is in direct contrast to results obtained from molecular phylogenetic surveys of BNR systems where most sludge 16S rDNA clones belong to subdivision 1 and only a few to subdivision 3. It is suggested that filamentous bacteria belonging to the Chloroflexi phylum account for a large fraction of phylogenetically uncharacterized filaments in BNR systems and are likely to be abundant in such systems on a global scale.
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nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA b
bThe GenBank accession number for the sequences of the tnpA-like gene, nahG and nahR of P. putida NCIB 9816-4 is AF491307. The GenBank accession numbers for the sequences of the nahR–nahG intergenic region and the nahR homologue genes of strains Cg1, Cg2, Cg5, Cg7, Cg9, Cg11, Hg8 and N1 are AF491308–AF491315, respectively. The GenBank accession numbers for the sequences of nahR from sediment-extracted DNA are AF491316–AF491324.
In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989) R33 . J Bacteriol 171, 837–846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5′-ATTCACGCTN2TGAT-3′). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.
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Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing c
cThe GenBank accession numbers for the sequences reported in this paper are AY080911–AY080961.
Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3·5) microcosms that were exposed to 13CH3OH or 13CH4. Distinct 13C-labelled DNA (13C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the 13C-DNA fractions, suggesting certain AOB assimilated a significant proportion of 13CO2, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the 13CH3OH and 13CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.
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Cloning, sequencing and expression of an α-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme a
More LessaThe GenBank accession number for the sequence reported in this paper is AF442961.
A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the α-amylase family and showed the greatest homology to the Bacillus megaterium group of α-amylases. The amyA gene was expressed in E. coli as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 °C in 5% (w/v) NaCl at pH 7·5, with significant activity retained in the presence of up to 25% (w/v) NaCl. It had a specific activity of 22·32 U mg−1 and required NaCl and CaCl2 for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H. orenii AmyA.
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Cross-talk between the L-sorbose and D-sorbitol (D-glucitol) metabolic pathways in Lactobacillus casei a
More LessaThe GenBank accession number for the sequence reported in this paper is AF396831.
A gene encoding sorbitol-6-phosphate dehydrogenase (SorF) belonging to the sorbose operon (sorFABCDG) has been characterized in Lactobacillus casei. Inactivation of this gene revealed the presence of another sorbitol-6-phosphate dehydrogenase that was induced by D-sorbitol (D-glucitol). The gene encoding this activity (gutF) has also been isolated, sequenced and disrupted. The sorbitol-6-phosphate dehydrogenase genes (sorF, gutF) were required for growth on L-sorbose and D-sorbitol, respectively. Biochemical and transcriptional analyses of the wild-type and mutant strains demonstrated that L-sorbose and D-sorbitol induced sorF and the gene encoding the sorbose operon activator (sorR), while the expression of gutF was only activated by D-sorbitol. Furthermore, these studies indirectly suggested that a common metabolite of the L-sorbose and D-sorbitol metabolic pathways (probably D-sorbitol 6-phosphate) would act as the effector of SorR. The same effector would also be the inducer of gutF, although the two pathways seem to be subject to distinct regulatory mechanisms.
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High-level resistance to class IIa bacteriocins is associated with one general mechanism in Listeria monocytogenes
Class IIa bacteriocins may be used as natural food preservatives, yet resistance development in the target organisms is still poorly understood. In this study, the understanding of class IIa resistance development in Listeria monocytogenes is extended, linking the seemingly diverging results previously reported. Eight resistant mutants having a high resistance level (at least a 103-fold increase in MIC), originating from five wild-type listerial strains, were independently isolated following exposure to four different class IIa bacteriocin-producing lactic acid bacteria (including pediocin PA-1 and leucocin A producers). Two of the mutants were isolated from food model systems (a saveloy-type sausage at 10 °C, and salmon juice at 5 °C). Northern blot analysis showed that the eight mutants all had increased expression of EIIBgl and a phospho-β-glucosidase homologue, both originating from putative β-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). However, disruption of these genes in a resistant mutant did not confer pediocin sensitivity. Comparative two-dimensional gel analysis of proteins isolated from mutant and wild-type strains showed that one spot was consistently missing in the gels from mutant strains. This spot corresponded to the MptA subunit of the mannose-specific PTS, , found only in the gels of wild-type strains. The mptACD operon was recently shown to be regulated by the σ54 transcription factor in conjunction with the activator ManR. Class IIa bacteriocin-resistant mutants having defined mutations in mpt or manR also exhibited the two diverging PTS expression changes. It is suggested here that high-level class IIa resistance in L. monocytogenes and at least some other Gram-positive bacteria is developed by one prevalent mechanism, irrespective of wild-type strain, class IIa bacteriocin, or the tested environmental conditions. The changes in expression of the β-glucoside-specific and the mannose-specific PTS are both influenced by this mechanism. The current understanding of the actual cause of class IIa resistance is discussed.
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Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium
More LessVanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation. Some degree of resistance to V is inducible by prior exposure to the metal. One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P. aeruginosa PA 59.20, a clinical isolate. This mutant had an insertion in a non-coding region, upstream of a cluster of four genes. Three of them show similarities to genes corresponding to known P. aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin. This cluster was named mexGHI-opmD. By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P. aeruginosa PAO1. Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing. All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster. The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay. Total extracts from the three mutants failed to induce the production of violacein by C. violaceum, although AHLs were detected by TLC and C. violaceum overlay. Violacein production was restored by complementation with mexGHI-opmD. The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell. In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P. aeruginosa.
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Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination
More LessThe location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB – in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme’s hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD600 fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore’s ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.
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Permeability of Coxiella burnetii to ribonucleosides
More LessKnowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete. The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes. Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities. Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides. Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine. Both phases were poorly active in cytidine uptake. Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil. Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism. Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process. It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.
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The signal transducer PII and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation a
aThe GenBank accession number for the glnB gene sequence reported in this paper is AJ271089.
The amino acid sequence of the signal transducer PII (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria. PII of P. marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942. Moreover, P. marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite. This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (Ci; K m,app 240 and 4 μM, respectively), a result consistent with the unphosphorylated form of PII acting as a sensor for the control of Ci acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803. The present data are discussed in relation to the genetic information provided by the P. marinus MED4 genome sequence.
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Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16
More LessRegulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the −10 region and a region immediately upstream of the −35 region of the σ70 promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the σ54-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaRΩKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed. In addition to this, phaR expression is subject to autoregulation. Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.
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A zinc metalloprotease inhibitor, Inh, from the insect pathogen Photorhabdus luminescens
More LessThe entomopathogen Photorhabdus luminescens secretes many proteins during the late stages of insect larvae infection and during in vitro laboratory culture. The authors have previously characterized and purified a 55 kDa zinc metalloprotease, PrtA, from culture supernatants of P. luminescens. PrtA is secreted via a classical type I secretory pathway and is encoded within the operon prtA–inh–prtBCD. The 405 bp inh gene encodes a 14·8 kDa pre-protein that is translocated to the periplasm by the classical signal-peptide-dependent sec pathway, yielding the mature 11·9 kDa inhibitor Inh. Inh is a specific inhibitor of the protease PrtA. This study describes the purification of Inh and the initial characterization of its in vitro protease inhibition properties.
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The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit
More LessProtein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase α subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the β subunit nor the N- and C-terminal regions of the α subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the α subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.
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The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion
More LessThe ferric uptake regulator (Fur) of Pseudomonas aeruginosa was expressed in Escherichia coli in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn2+ that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn2+ ion per monomer. Thus, the P. aeruginosa Fur can probably accommodate a single Zn2+ ion bound to the metal-sensing site. The single cysteine residue of P. aeruginosa Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the E. coli protein, and is believed to provide one of the ligands to a structural Zn2+ ion. This cysteine residue was shown to be dispensable for the in vivo activity of P. aeruginosa Fur, which is consistent with the suggestion that the P. aeruginosa protein does not contain a structural Zn2+ ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of P. aeruginosa Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable.
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Regulation of polyphenol oxidase activities and melanin synthesis in Marinomonas mediterranea: identification of ppoS, a gene encoding a sensor histidine kinase a
More LessaThe GenBank accession number for the sequence reported in this paper is AF398464.
Marinomonas mediterranea is a melanogenic marine bacterium that expresses two different polyphenol oxidases. One of them is a multipotent laccase able to oxidize a wide range of substrates. The second enzyme is an SDS-activated tyrosinase. Using transposon mutagenesis, a mutant affected in the regulation of both polyphenol oxidase activities and melanogenesis has been isolated. The sequencing of the gene disrupted by the mini-Tn10 transposon in this mutant indicates that it encodes a hybrid sensor kinase. This sensor kinase shows three phosphorylated conserved domains: the transmitter domain containing a histidine site typical of sensor kinases, a receiver domain with an aspartate residue and an additional phosphotransferase domain with a second conserved histidine. This structural organization is characteristic of kinases participating in a phosphorelay system. Northern blot and lacZ operon fusions indicate that the multipotent laccase activity is regulated not only by PpoS but also by growth phase at the transcriptional level. These results suggest that PPO activities and melanin synthesis play a role in the adaptive response of M. mediterranea to stressful environmental conditions.
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Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis
Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, Trichomonas vaginalis. Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD+-dependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH:ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant Trichomonas vaginalis compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.
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Plasmid-borne macrolide resistance in Micrococcus luteus a
More LessaThe GenBank accession number for the sequence reported in this paper is AF462611.
A plasmid designated pMEC2 which confers resistance to erythromycin, other macrolides, and lincomycin was detected in Micrococcus luteus strain MAW843 isolated from human skin. Curing of this approximately 4·2 kb plasmid from the host organism resulted in erythromycin sensitivity of the strain. Introduction of pMEC2 into a different M. luteus strain conferred erythromycin resistance upon this strain. Macrolide resistance in M. luteus MAW843 was an inducible trait. Induction occurred at subinhibitory erythromycin concentrations of about 0·02–0·05 μg ml−1. Erythromycin and oleandomycin were inducers, while spiramycin and tylosin exerted no significant inducer properties. With heterologous expression experiments in Corynebacterium glutamicum, using hybrid plasmid constructs and deletion derivatives thereof, it was possible to narrow down the location of the plasmid-borne erythromycin-resistance determinant to a region of about 1·8 kb of pMEC2. Sequence analysis of the genetic determinant, designated erm(36), identified an ORF putatively encoding a 281-residue protein with similarity to 23S rRNA adenine N 6-methyltransferases. erm(36) was most related (about 52–54% identity) to erythromycin-resistance proteins found in high-G+C Gram-positive bacteria, including the (opportunistic) pathogenic corynebacteria Corynebacterium jeikeium, C. striatum, C. diphtheriae and Propionibacterium acnes. This is believed to be the first report of a plasmid-borne, inducible antibiotic resistance in micrococci. The possible role of non-pathogenic, saprophytic micrococci bearing antibiotic-resistance genes in the spreading of these determinants is discussed.
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Differential effects of Kid toxin on two modes of replication of lambdoid plasmids suggest that this toxin acts before, but not after, the assembly of the replication complex
More LessKid is a small protein that is encoded by plasmid R1. It is a toxin that belongs to a killer system that ensures the stability of the plasmid in host cells. The results of previous studies have suggested that Kid is an inhibitor of DNA replication, possibly acting at the onset of initiation. Here, the authors tested the effects of Kid on oriλ-intitiated and oriJ-initiated replication, which may be driven by both the newly assembled replication complex and the heritable complex. It was found that Kid inhibits only replication that is driven by the newly assembled replication complex. The authors also report that Kid inhibits ColE1-like plasmid replication in vivo, in agreement with the previously reported inhibition of ColE1 during in vitro replication. It is proposed that the Kid toxin acts at the level of replication either by preventing de novo assembly of the replication complex or by impairing the functional interactions of the replication complex at the initiation stage.
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Diversity within inc genes of clinical Chlamydia trachomatis variant isolates that occupy non-fusogenic inclusions a
More LessaThe GenBank accession numbers for the sequences reported in this paper can be found in Fig. 1 F1 .
The obligately intracellular chlamydiae are bacterial pathogens that occupy intracellular vacuoles, termed inclusions, as they develop and multiply. Typical Chlamydia trachomatis isolates occupy inclusions that fuse with other C. trachomatis inclusions within cells infected with multiple elementary bodies (wild-type phenotype). The authors of this study have recently described C. trachomatis isolates that form multiply-lobed, non-fusogenic inclusions within single cells infected with multiple elementary bodies (variant phenotype). Inclusions formed by these isolates uniformly lacked the protein IncA on the inclusion membrane (IM). In the present work, the study of the C. trachomatis inclusion phenotype has been expanded to include 27 variant and 13 wild-type isolates. Twenty-four of the 27 variant isolates were IncA-negative, as detected by fluorescence microscopy and immunoblotting, but three variants localized IncA to the IM. The IncA-positive variants formed inclusions that fused, at a reduced rate, with those occupied by wild-type isolates and with inclusions formed by other IncA-positive variants. Nucleotide-sequence analysis of the incA sequences from the variant isolates identified a variety of distinct sequence polymorphisms relative to incA from wild-type strains. The authors also demonstrate that a second Inc protein, CT223p, is not found in the IM in selected C. trachomatis isolates. No change in the structure or the fusogenicity of the inclusions was associated with the presence or absence of CT223p.
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BfpU, a soluble protein essential for type IV pilus biogenesis in enteropathogenic Escherichia coli
A cluster of 14 genes located on the large plasmid of enteropathogenic Escherichia coli (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant E. coli host. The fifth gene in the cluster, bfpU, encodes a protein that is predicted to be localized to the periplasmic space. To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar bfpU mutant EPEC strain by allelic exchange. The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP. These phenotypes were restored to the bfpU mutant by a plasmid containing bfpU. There was no difference between the wild-type and bfpU mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes. Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm. Thus, BfpU represents a novel protein required for type IV pilus assembly.
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Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression a
aThis paper is dedicated to the memory of our dear friends and colleagues Giuseppe Carruba and Franco Tatò, who died prematurely.
bThe GenBank accession number for the sequence reported in this paper is AJ315184.
In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB–apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2·2±0·3 min, versus 27±4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB–apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB–apy transcript.
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Evolution of multi-gene segments in the mutS–rpoS intergenic region of Salmonella enterica serovar Typhimurium LT2 a
aThe GenBank accession number for the sequence reported in this paper is AY050714.
The nucleotide sequence of the 12·6 kb region between the mutS and rpoS genes of Salmonella enterica serovar Typhimurium LT2 (S. typhimurium) was compared to other enteric bacterial mutS–rpoS intergenic regions. The mutS–rpoS region is composed of three distinct segments, designated HK, O and S, as defined by sequence similarities to contiguous ORFs in other bacteria. Inverted chromosomal orientations of each of these segments are found between the mutS and rpoS genes in related Enterobacteriaceae. The HK segment is distantly related to a cluster of seven ORFs found in Haemophilus influenzae and a cluster of five ORFs found between the mutS and rpoS genes in Escherichia coli K-12. The O segment is related to the mutS–rpoS intergenic region found in E. coli O157:H7 and Shigella dysenteriae type 1. The third segment, S, is common to diverse Salmonella species, but is absent from E. coli. Despite the extensive collinearity and conservation of the overall genetic maps of S. typhimurium and E. coli K-12, the insertions, deletions and inversions in the mutS–rpoS region provide evidence that this region of the chromosome is an active site for horizontal gene transfer and rearrangement.
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Protein signatures distinctive of chlamydial species: horizontal transfers of cell wall biosynthesis genes glmU from archaea to chlamydiae and murA between chlamydiae and Streptomyces a
More LessaThe GenBank accession numbers for the sequences reported in this paper are indicated in the text.
Chlamydiae are major human and animal pathogens. Based on alignments of different protein sequences, a number of conserved indels (insertion/deletions) were identified that appear to be unique and distinctive characteristics of the chlamydial species. The identified signatures include one 16 aa and two single aa inserts in the enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA), a 1 aa insert in protein synthesis elongation factor P (EF-P), a 1 aa insert in the Mg2+ transport protein (MgtE), a 1 aa insert in the carboxy-terminal protease and a 1 aa deletion in the tRNA (guanine-N 1-)-methyltransferase (TrmD) protein. The homologues of these proteins are found in all major groups of bacteria and the observed indels are present in all available chlamydial sequences but not in any other species (except for the large insert in MurA in Streptomyces). The validity of three of these signatures (MurA, EF-P and MgtE) was tested by PCR amplifying the signature regions from several chlamydial species for which no sequence information was available. All Chlamydiaceae species for which specific fragments could be amplified (Chlamydia suis, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila felis) contained the expected signatures. Additionally, a fragment of the murA gene from Waddlia chondrophila and the efp gene from Simkania negevensis, two chlamydia-like species, were also cloned and sequenced. The presence of respective indels in these species provides strong evidence that they are specifically related to the traditional chlamydial species, and that these signatures may be distinctive of the entire Chlamydiales order. A 17 aa conserved indel was also identified in the cell wall biosynthesis enzyme UDP-N-acetylglucosamine pyrophosphorylase (GlmU), which is shared by all archaeal and chlamydial homologues. The gene for this protein is indicated to have been horizontally transferred from an archaeon to a common ancestor of the chlamydiae. The results also support a lateral transfer of the murA gene between chlamydiae and Streptomyces. The large inserts in these peptidoglycan synthesis related genes in chlamydiae could account for their unusual cell-wall characteristics. These signatures are also potentially useful for screening of the chlamydiae species.
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Small genome of Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies irreversible specialization to an intracellular lifestyle a
More LessaThe GenBank accession number for the sequence reported in this paper is AF495758.
Blochmannia (Candidatus Blochmannia gen. nov.) is the primary bacterial endosymbiont of the ant genus Camponotus. Like other obligate endosymbionts of insects, Blochmannia occurs exclusively within eukaryotic cells and has experienced long-term vertical transmission through host lineages. In this study, PFGE was used to estimate the genome size of Blochmannia as approximately 800 kb, which is significantly smaller than its free-living relatives in the enterobacteria. This small genome implies that Blochmannia has deleted most of the genetic machinery of related free-living bacteria. Due to restricted gene exchange in obligate endosymbionts, the substantial gene loss in Blochmannia and other insect mutualists may reflect irreversible specialization to a host cellular environment.
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Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium a
More LessaThe GenBank accession numbers for the nifD sequences determined in this work are AF484254–AF484287.
Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase α-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.
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Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA
Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated. Priming with oligo(dT) yielded more cDNA than priming with an arbitrary primer for only 1 of 12 unrelated M. tuberculosis genes, as measured by competitive PCR. Priming with oligo(dT) yielded cDNA for only 30% of the genes primed for by 37 M. tuberculosis genome-directed oligonucleotides, as assessed by hybridization of cDNA with an M. tuberculosis microarray. These data demonstrate that priming of reverse transcription of mycobacterial mRNA with oligo(dT) does not yield representative samples of cDNA.
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Silencing of the Escherichia coli bgl operon by RpoS requires Crl
More LessSilencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS. Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter. However, the expression level of activated bgl operon derivatives varies with the strain background. Here it is shown that the repression of the bgl operon by RpoS requires Crl. Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes. In a Crl-negative strain RpoS had no effect on the bgl operon. The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E. coli strains. Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.
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The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris a
More LessaThe GenBank accession number for the sequence reported in this paper is AB066287.
The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C. maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5′-untranslated regions (TS1 and TS2) were present, of which the 5′ end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.
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Identification and characterization of novel small RNAs in the aspS–yrvM intergenic region of the Bacillus subtilis genome
A novel RNA species was isolated from Bacillus subtilis, and its sequence was determined and mapped to its genetic position. This RNA was termed BS190 RNA from the length of its mature form (190 nt), and the gene encoding it is located within the aspS–yrvM intergenic region of the B. subtilis genome. Northern blotting revealed that the novel RNA species is transcribed in vegetative cells as a larger precursor (BS201 RNA, 201 nt). After transcription, the 5′ end of the precursor is processed to generate the mature form, BS190 RNA. A computer-aided prediction of the secondary structure of BS190 RNA showed that it can be folded into a single hairpin structure with some bulge structures. The authors found that the growth rate of a ΔBS190 mutant strain of B. subtilis was reduced when compared to the wild-type. A phylogenetic comparison of the sequence of the BS190 RNA gene with sequences from the databases suggests that RNA related to BS190 RNA appears to be encoded in the genomes of Bacillus halodurans and Listeria monocytogenes.
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Protective role of trehalose during severe oxidative stress caused by hydrogen peroxide and the adaptive oxidative stress response in Candida albicans
More LessThe cellular response to the oxidative stress caused by hydrogen peroxide and its putative correlation with the stress protector trehalose was investigated in Candida albicans CAI.4 and the tps1/tps1 double mutant, which is deficient in trehalose synthesis. When exponential wild-type blastoconidia were exposed to high concentrations of hydrogen peroxide, they displayed a high cell survival, accompanied by a marked rise of intracellular trehalose. The latter is due to a moderate activation of trehalose synthase and the concomitant inactivation of neutral trehalase. Identical challenge in the tps1/tps1 double mutant severely reduced cell viability, a phenotype which was suppressed by overexpression of the TPS1 gene. Pretreatment of growing cultures from both strains with either a low, non-lethal concentration of H2O2 (0·5 mM) or a preincubation at 37 °C, induced an adaptive response that protected cells from being killed by a subsequent exposure to oxidative stress. During these mild oxidative preincubations, trehalose was not induced in CAI.4 cells and remained undetectable in their tps1/tps1 counterpart. Blastoconidia from the two strains exhibited a similar degree of cell protection during the adaptive response. The induction of trehalose accumulation by H2O2 was not due to an increased expression of TPS1 mRNA. These results are consistent with a mainly protective role of trehalose in C. albicans during direct oxidative stress but not during acquired oxidative tolerance.
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A PCR-based strategy to generate integrative targeting alleles with large regions of homology
Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.
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Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant a
More LessaThe GenBank accession numbers for the sequences reported in this paper are AY035556 and AF489498.
The Cryptococcus neoformans MET3 cDNA (encoding ATP sulfurylase) was cloned by complementation of the corresponding met3 mutation in Saccharomyces cerevisiae. Sequence analysis showed high similarity between the deduced amino acid sequence of the C. neoformans Met3p and other fungal ATP sulfurylases. A C. neoformans met3 mutant was made by targeted insertional mutagenesis, which had the expected auxotrophic phenotype, and reconstituted the met3 mutant to Met+. In vitro, the C. neoformans met3 mutant had a substantial defect in melanin formation, significantly reduced growth rate, and greatly increased thermotolerance. In the murine inhalation infection model, the met3 mutant was avirulent and was deficient in its ability to survive in mice. It is concluded that, in contrast to the yeast form of Histoplasma capsulatum, in C. neoformans the sulfate-assimilation arm of the methionine biosynthetic pathway plays an important role in vitro, even in the presence of abundant exogenous methionine, and is critical for virulence, and indeed for survival, in vivo.
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cAMP alteration of growth rate of Aspergillus fumigatus and Aspergillus niger is carbon-source dependent
More LesscAMP signalling has been shown to be essential for normal growth, morphology and virulence in fungal pathogens of both plants and animals. The effects of exogenous cAMP on the growth of the opportunistic pathogen Aspergillus fumigatus were compared to those of Aspergillus niger, which has previously been demonstrated to respond to extracellular cAMP. Both cAMP and phosphodiesterase inhibitors markedly reduced the radial growth rate of A. niger after 48 h on minimal medium with glucose as the carbon source, whereas the growth of A. fumigatus was not affected by cAMP. However, when glycerol, which does not initiate carbon catabolite repression, was used as a carbon source, cAMP inhibited the radial growth rate of only A. fumigatus (P<0·05). The addition of cAMP to glycerol-minimal medium resulted in a fourfold increase in protein kinase A activity in A. fumigatus cell extracts when compared with pre-treatment samples. The protein kinase A activity in A. fumigatus cell extracts from cultures grown in glucose did not change significantly with the addition of cAMP. These studies demonstrate that although the growth rates of both A. fumigatus and A. niger are sensitive to the addition of exogenous cAMP, the response of each organism is distinct and dependent on the carbon source used.
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Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger a
aThe EMBL accession number for the sequence reported in this paper is AJ296317.
The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the ΔpkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.
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